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ABSTRACT: Double-stranded RNA interference can be used to silence gene expression in various organisms. Sustained RNAi-mediated gene
silencing is typically triggered by hairpin RNAs (hpRNAs) generated by the transcription of inverted repeat (IR) DNA constructs.
In this paper, we describe a transient expression assay that uses a green fluorescent protein marker gene (GFP) fused to the
coat protein gene (CP) of papaya ring spot virus. This yields a nuclear and cytomembrane localized protein expression vector that can be used to
monitor the silencing efficiency of inverted repeats. An ihpRNA was derived from a 215nt IR segment of the 3′ end of the
CP gene and recombined into the pHellsgate12 vector. We also recombined the 861nt CP into the destination vectors pHellsgate12 and pWatergate using the GatewayTM technology. Nicotiana benthamiana protoplasts were co-transfected with (1) pGA, (2) pGA+pHellsgate12-CP
IR, (3) pGA+pWatergate-CP
IR, and (4) pGA+pHellsgate12-CP
215IR. Expression of the GFP fusion protein decreased by 77.7, 75.4, and 65.6% for the three constructs. Transfection with (2) and (3) yielded a silencing
efficiency significantly higher than that of the (4) (P<0.05), as measured by fluorescence intensity upon co-transfection,
using the vector pGA-DsRed as an internal control. Depletions of 90.7, 91.5, and 87.5% of the CP transcript were observed by RT-PCR in protoplasts co-transfected with the (1) pGA, (2) pGA+pHellsgate12-CPIR, and (3) pGA+pWatergate-CPIR, (4) pGA+pHellsgate12-CP
215IR. Transient RNAi depletion of the target polypeptide was also detected by western blot analysis. Short RNA fragments complementary
to the p6a and p7a antisense probes were detected by Ribonuclease Protection Assays.
Plant Cell Tissue and Organ Culture 100(2):139-148. · 3.63 Impact Factor